Cascade continuous fermentation of cellulosic biomass by integration bioprocessing

Abstract

A system for converting biomass to ethanol and other desired products is disclosed. The system comprises a plurality of bioreactors connected in series, and the bioreactors are connected through mills. The configuration enables the reaction time to be shortened, which is beneficial to industrial processing. The present disclosure relates to systems and methods for efficient conversion of biomass using integrated bioprocessing (CBP) to convert cellulosic biomass to fuels and / or chemicals, without the need for addition of enzymes and without the need for thermochemical pretreatment.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 399,133, filed on August 18, 2022, the entire contents of which are incorporated herein by reference.

[0003] Government Rights

[0004] This invention was made with government support from the Center for Bioenergy Innovation (CBI), a U.S. Department of Energy (DOE) Bioenergy Research Center supported by the Office of Biological and Environmental Research in the Office of Science. The government has certain rights in this invention. Technical Field

[0005] The present invention relates to a system for converting biomass into biofuel or other useful products. Background Art

[0006] Biomass is a relatively cheap, renewable and abundant material that can be used to produce fuels, chemicals, fibers and energy. However, the large-scale utilization of plant biomass is at least partially hindered by the lack of technology that can efficiently convert biomass into component parts or active intermediates at low cost. For example, most plant biomass is resistant to digestion by cellulase. In addition, since obtaining sugars in lignocellulose requires expensive enzymes and bioprocessing equipment, the development of producing biofuels from non-food biomass is slow. Summary of the invention

[0007] The present disclosure relates to systems and methods for efficient conversion of biomass using a system including multiple reactors connected in series and using consolidated bioprocessing (CBP) to convert cellulosic biomass into fuels and / or chemicals without the addition of enzymes and without thermochemical pretreatment.

[0008] Thermophilic, lignocellulose fermenting bacteria have the potential to significantly reduce the cost of producing renewable biofuels from cellulosic biomass. However, challenges remain to make thermophilic bacteria practical. Robust industrial biocatalysts with the following characteristics are needed to improve processing: no reduction in solubilization at high solids content, complete utilization of hemicellulose solubilization products, production of high product titers. Energy-saving means to improve accessibility to biological attack will also help improve the economics of biomass utilization.

[0009] To this end, integrated bioprocessing (CBP) using thermophilic bacteria needs to be performed in (multiple) bioreactors that need to be configured for maximum effectiveness to achieve cost-effectiveness. In certain embodiments, the present disclosure provides a system with multiple continuous, well-mixed bioreactors connected in series - a configuration known as "cascade continuous".

[0010] In certain embodiments, the cascade continuous design helps to reduce the reaction time, which, in turn, helps to reduce processing costs. Continuous processing provides special advantages for processing lignocellulose by CBP. Compared with the more traditional processing strategies involving the addition of cellulase and related enzymes, some of these advantages are surprising and unexpected. In certain embodiments, the first-order kinetics of many microbial systems that mediate lignocellulose deconstruction have been observed, including the limited culture of thermophilic bacteria. Analysis based on first-order kinetics shows that in a single-stage well-mixed continuous bioreactor, the reaction time to reach a feasible degree of substrate solubilization is about 4.5 days. This reaction time is very similar to the reaction time observed in the batch culture of thermophilic bacteria fermenting lignocellulose. In contrast, based on the same analysis for predicting a 4.5-day reaction time in a single-stage CSTR, the required reaction time for three bioreactors arranged in series is only 1.8 days (reduced by 2.5 times).

[0011] In some embodiments, a system for converting biomass into liquid fuels is disclosed, the system comprising: a) a liquid reservoir; and b) a plurality of reactors arranged in series. In one aspect, each reactor has an inlet and an overflow outlet, wherein the inlet of a first reactor of the plurality of reactors is connected to the liquid reservoir. In another aspect, the plurality of reactors comprises a defined culture comprising at least one living organism capable of producing at least one carbohydrate active enzyme (CAZyme).

[0012] Here, biomass moves from one reactor to the next through an overflow outlet, and the steps of CAZyme production, substrate hydrolysis and fermentation by the defined culture without added enzymes all occur within each of the plurality of reactors. The enzymes (i.e., CAZymes) are produced by the living organisms. No other enzymes are added to the reactors in series, a feature that is contrary to the conventional processing paradigm of batch operation, which typically adds enzymes during processing to increase the enzyme: substrate ratio.

[0013] In some embodiments, the system is configured such that overflow from the first reactor in the series is fed to the second reactor immediately downstream of the first reactor, and overflow from the Nth reactor is fed to the N+1th reactor immediately downstream of the Nth reactor, and so on, where N is an integer from 1 to n, and where n is an integer, and n+1 is equal to the total number of reactors in the plurality of reactors.

[0014] In some embodiments, the system may further include one or more mills placed between any two adjacent reactors to further process the moving biomass.

[0015] In some embodiments, the system can include a starting biomass, wherein the starting biomass is loaded into the reservoir, and the biomass flows unidirectionally from the reservoir to the first reactor.

[0016] In some embodiments, the plurality of reactors may include 3 or more reactors, or at least 4 reactors, or at least 5 reactors, or the like.

[0017] In some embodiments, the system operates continuously or semi-continuously. On the one hand, in a CBP process using the disclosed system, the initial enzyme concentration in the initial bioreactor in the cascade is high, and the available substrate concentration is high, which is advantageous compared to a batch or single-stage CSTR configuration. On the other hand, a series of reactors operating continuously or semi-continuously significantly shortens the reaction time from biomass to liquid fuel compared to the reaction time in a batch or single-stage continuous bioreactor.

[0018] In some embodiments, the ratio between the CAZyme and the substrate in the first reactor is at least 0.1 or 1 unit of CAZyme, or at least 10 units of CAZyme per pmol of the substrate. In some embodiments, the ratio between the CAZyme and the substrate in each reactor of the system is at least 0.1 or 1 unit of CAZyme, or at least 10 units of CAZyme per pmol of the substrate. In some embodiments, CAZyme is in excess of the substrate to ensure utilization of the substrate and fast reaction time.

[0019] In some embodiments, the liquid fuel is ethanol or other chemicals.

[0020] In some embodiments, the system can include a defined culture comprising a living organism, such as Clostridium thermocellum, and a hemicellulose fermenting microorganism selected from the group consisting of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium saccharolyticum.

[0021] In some embodiments, the starting biomass is one selected from the group consisting of corn stover, sugar cane, wheat straw or rice straw, miscanthus, switchgrass, energy cane, and combinations thereof.

[0022] In some embodiments, the cascade continuous system is applied to CBP instead of simultaneous saccharification and fermentation (SSF) because the benefits of the cascade continuous configuration are limited to CBP and not applicable to systems with added enzymes, including but not limited to SSF. The traditional CBP process usually has a low initial enzyme concentration, while the cascade continuous system using CBP can achieve a higher initial enzyme concentration, which helps to accelerate the conversion of biomass to biofuels.

[0023] In some embodiments, a method for producing liquid fuel from biomass using the above system is also disclosed. The method may include: a) loading the starting biomass into a reservoir; b) moving the biomass from the reservoir to a first reactor, in which at least a portion of the biomass is converted into alcohol; c) operating the system continuously or semi-continuously, wherein the reservoir feeds the first reactor, and the overflow from the Nth reactor moves to the N+1th reactor immediately downstream of the Nth reactor; d) repeating the above steps (a)-(c) so that the biomass moves from the Nth reactor to the N+1 reactor through the reactors in series until it reaches the last reactor; e) operating the system continuously or semi-continuously, wherein the steps of CAZyme production, substrate hydrolysis and fermentation of the defined culture without the addition of enzymes all occur in each of the multiple reactors; and f) collecting the liquid fuel from the multiple reactors.

[0024] In some embodiments, the solids loading in the system is about 5-30 wt.%, or about 10-25 wt.% solids, or about 15-25 wt.% solids. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A preferred embodiment of the configuration of the bioreactor described herein is shown.

[0026] Figure 2 Comparison of solubilization data using fungal cellulases SSF and C. thermocellum is shown.

[0027] Figure 3 Example graphs showing the kinetic characteristics of continuous cultures. DETAILED DESCRIPTION

[0028] This article describes systems and methods for converting biomass into ethanol or other desired products. In a preferred embodiment, for example, Figure 1 As shown, the system of the present invention includes: a) a liquid reservoir; b) a first bioreactor, a second bioreactor, and a third bioreactor; and c) a first mill coupled to the first bioreactor and the second bioreactor and a second mill coupled to the second bioreactor and the third bioreactor. In other embodiments, the system includes: a) a liquid reservoir; b) a plurality of bioreactors, wherein each bioreactor is connected in series; and c) a plurality of mills, wherein each mill is coupled to two bioreactors.

[0029] The configuration of the system described herein confers several important advantages: 1) conditions favor microbial growth and rapid reaction in the initial phase, and favor solubilization of a high proportion of carbohydrates and product titer in the later phase; 2) reaction times are shortened several-fold compared to batch or single-stage continuous processing. This benefit is unique to CBP and is not achieved by more traditional processes with enzyme addition; 3) continuous fermentation is beneficial to C-CBP because it avoids mixing and mass transfer challenges at high solids content and is the preferred mode of inoculum preparation for obligate anaerobes.

[0030] The embodiments of the present disclosure are further illustrated by the following items.

[0031] Item 1. A system for converting biomass into liquid fuel, comprising:

[0032] a) a liquid reservoir; and

[0033] b) a plurality of reactors arranged in series, each reactor having an inlet and an overflow outlet, the inlet of a first reactor of the plurality of reactors being connected to the liquid reservoir,

[0034] wherein said plurality of reactors comprises a defined culture comprising at least one living organism capable of producing at least one carbohydrate active enzyme (CAZyme), wherein the steps of CAZyme production, substrate hydrolysis and fermentation by said defined culture without added enzymes all occur within each of said plurality of reactors.

[0035] Item 2. A system according to Item 1, wherein the overflow from the first reactor in the series is fed to the second reactor immediately downstream of the first reactor, and the overflow from the Nth reactor is fed to the N+1th reactor immediately downstream of the Nth reactor, wherein N is an integer from 1 to n, and wherein n is an integer, and n+1 is equal to the total number of reactors in the multiple reactors.

[0036] Item 3. The system according to any of the preceding items, further comprising one or more mills placed between two adjacent reactors.

[0037] Item 4. The system according to any of the preceding items, further comprising a starting biomass, wherein the starting biomass is loaded into the reservoir, and the biomass flows unidirectionally from the reservoir to the first reactor.

[0038] Item 5. A system according to any preceding item, wherein the plurality of reactors comprises 3 or more reactors.

[0039] Item 6. A system according to any preceding item, wherein the system operates continuously or semi-continuously.

[0040] Item 7. The system according to any of the preceding items, wherein the continuous or semi-continuous operation of the series of reactors shortens the reaction time from the biomass to the liquid fuel.

[0041] Item 8. A system according to any preceding item, wherein the reaction time from the biomass to the liquid fuel is shorter than the reaction time in a batch or single-stage continuous bioreactor.

[0042] Item 9. A system according to any preceding item, wherein the ratio between the CAZyme and the substrate in the first reactor is at least one unit of CAZyme per pmol of the substrate.

[0043] Item 10. A system according to any preceding item, wherein the liquid fuel is ethanol.

[0044] Item 11. A system according to any preceding item, wherein the defined culture comprises Clostridium thermocellum and a hemicellulose fermenting microorganism selected from the group consisting of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium saccharolyticum.

[0045] Item 12. A system according to any preceding item, wherein the starting biomass is one selected from the group consisting of corn stover, sugar cane, wheat straw or rice straw, miscanthus, switchgrass, energy cane, and combinations thereof.

[0046] Item 13. A method for producing liquid fuel from biomass using the system of claim 1, comprising:

[0047] a) loading the starting biomass into a reservoir,

[0048] b) moving the biomass from the reservoir to a first reactor wherein at least a portion of the biomass is converted to alcohol,

[0049] c) operating the system continuously or semi-continuously, wherein the liquid reservoir feeds the first reactor and overflow from the Nth reactor moves to the N+1th reactor immediately downstream of the Nth reactor,

[0050] d) repeating the above steps (a)-(c) so that the biomass moves from the Nth reactor to the N+1 reactor through the reactors in series until it reaches the last reactor,

[0051] e) operating the system continuously or semi-continuously, wherein the steps of CAZyme production by the defined culture without added enzymes, substrate hydrolysis and fermentation all occur within each of the reactors of the plurality of reactors, and

[0052] f) collecting the liquid fuel from the plurality of reactors.

[0053] Item 14. A method according to Item 13, wherein the liquid fuel is ethanol.

[0054] Item 15. A method according to any one of Items 13-14, wherein one or more mills placed between the reactor and an immediately downstream reactor mechanically process the overflow.

[0055] Item 16. A method according to any one of Items 13-15, wherein the system operates in a continuous or semi-continuous mode rather than in an intermittent mode.

[0056] Item 17. The method of any one of items 13-16, wherein the solid loading is about 10-25 wt. % solids.

[0057] Item 18. A method according to any one of Items 13-17, wherein the starting biomass is selected from the group consisting of corn stover, sugar cane, wheat straw or rice straw, miscanthus, switchgrass, energy sugar cane, and combinations thereof.

[0058] Item 19. A method according to any one of Items 13-18, wherein the ratio between the CAZyme and the substrate in the first reactor is at least one unit of CAZyme per pmol of the substrate.

[0059] For the purposes of this disclosure, the term "biomass" refers to cellulosic biomass. In one embodiment of the present disclosure, biomass refers to plant biomass, which includes any plant-derived material (woody or non-woody) provided on a sustainable basis. Plant biomass may include, but is not limited to, crop wastes and residues (such as corn stalks, wheat stalks, rice stalks, bagasse, etc.), grass crops (such as switchgrass, alfalfa, winter rye, etc.). Plant biomass may also include, but is not limited to, wood energy crops, wood wastes and residues (such as trees, softwood forest thinnings, bark wastes, sawdust, paper and pulp industry residues or waste liquids, wood fiber, etc.). In urban areas, plant biomass may include yard waste (such as grass trimmings, leaves, tree trimmings, firewood branches, etc.), vegetable processing wastes, and recycled cardboard and paper products.

[0060] Example

[0061] Kinetics and reactor design for rapid processing via C-CBP

[0062] In batch culture, usually 5 to 7 days are needed to achieve complete fermentation by CBP, which is indicated by stopping the production of gas. For industrial processing, shortening the reaction time is highly desirable. For a medium-sized facility producing 5,000,000 gallons of ethanol per year, bioconversion requires many large bioreactors (e.g., 15 to 20 reactors of 1,000,000 gallons). Therefore, bioreactors account for a large part in the total capital investment for any lignocellulose conversion processing, and are the largest cost factor of the integrated bioprocessing (consolidated bioprocessing with cotreatment, C-CBP) of co-treatment.

[0063] It has been widely observed that the reaction time for lignocellulose conversion is much longer compared to other plant-based feedstocks, which results in, for example, larger bioreactor volumes and investments. Table 1 summarizes the reaction times for lignocellulose conversion compared to other plant-based feedstocks.

[0064] Table 1

[0065]

[0066] 1 Communication with Brazilian producers and experts.

[0067] 2 Stowers et al., 2009. Confirmed through communication with industrial manufacturers

[0068] 3 Typical values ​​from the literature for both conventional and thermochemical pretreatment / fungal cellulase processing paradigms and C-CBP

[0069] For lignocellulose, the upper limit of ethanol titer is about 50 g / L due to material handling limitations. Shortening the reaction time is the most promising way to reduce bioreactor investment. However, this may receive little attention because it is believed to be limited by recalcitrance.

[0070] Continuous (or semi-continuous) processing of lignocellulose is one approach to address this problem. Although unreacted lignocellulose is not practical for mixing at the desired loading (>15 wt.%) of solids, biomediated deconstruction results in dramatic liquefaction (viscosity reduction by several orders of magnitude). In addition, continuous lignocellulose-grown cultures have significant advantages over batch cultures in terms of kinetic characterization. Industrial preparation of inoculum is easier with facultative aerobic bacteria (e.g., yeast, E. coli) than with the obligate anaerobes envisioned for C-CBP.

[0071] However, continuous (or semi-continuous) processing of lignocellulose presents several challenges. Few potential contaminants can compete with C. thermocellum (and similar microorganisms) for access to lignocellulose surfaces. In addition, wild-type thermophiles cannot tolerate the high ethanol concentrations that engineered strains need to produce in order to achieve commercial viability.

[0072] The descriptive framework for the rate of lignocellulose solubilization applicable to mixed enrichments and defined cultures is described by the following equation:

[0073]

[0074] Where: C 0is the carbohydrate load added to the bioreactor (g / L); C is the residual carbohydrate in the steady-state bioreactor (g / L); θ is the residence time (days); r is the carbohydrate solubilization rate (g / L*day); k is the solubilization rate constant (1 day -1 ),equal Figure 3 The slope of R is the fraction of recalcitrant carbohydrates, equal to Figure 3 The intercept of .

[0075] 2 days -1 The rate constants of explore the effects of continuous conversion of cellulosic biomass. Steady-state mass balance of carbohydrates with first-order rate constants and equal influx and efflux:

[0076]

[0077] where θ is the residence time (bioreactor working volume / volume flow rate.

[0078] The total carbohydrate concentration C is equal to the available carbohydrate concentration C * Add the unavailable concentration f r C o Sum:

[0079] C=C * +f r C 0

[0080] Substituting into the material balance, we obtain the following equation:

[0081]

[0082] This can be done with C * / C * 0 and θ to solve:

[0083]

[0084]

[0085] Considering the continuous lignocellulose fermentation with the kinetics described above and the following parameters: k = 2.0 day -1 , f r =0.1, (Given f r =0.1, C o =111). Taking these values ​​into account, the residence time to solubilize 90% of the available carbohydrates is considered:

[0086]

[0087] Single-stage steady-state CSTR:

[0088]

[0089] Five-stage CSTR in series: n = 5, residence time of each CSTR θ = 0.6 days

[0090] For each reactor: θ = 0.6 days, k = 2 days -1 , the results are summarized in Table 2 below:

[0091] Table 2 Summary of results of 5-stage CSTR in series

[0092]

[0093] For the limiting case of infinite-order CSTR:

[0094]

[0095] for The results are summarized in Table 3 below:

[0096] Table 3 Summary of infinite CSTR results

[0097] Reactor configuration Time to reach X*=0.9 (days) Level 1 CSTR 4.5 3-stage CSTR in series 1.8 Infinite-stage CSTR in series 1.15

[0098] The configuration of the bioreactor is extremely important and is a high-impact target for innovation. Microbial deconstruction has the potential to be much faster than current technology (which requires 5 to 7 days). This analysis does not take into account several key factors, including: the kinetics of high solids production; physiological effects; and the effects of possible acceleration from co-processing. The following summarizes the kinetic benefits of cascaded continuous operation, which are clearly unique to CBP:

[0099] Table 4 Summary of the benefits of cascade continuous operation

[0100]

[0101] Those skilled in the art will readily appreciate that suitable equivalents may be used to modify the systems and methods described herein and to substitute without departing from the scope of the embodiments disclosed herein. Certain embodiments have now been described in detail, which will be more clearly understood by reference to the following examples, which are for illustrative purposes only and are not intended to be limiting.

[0102] The contents of all references (including literature references, patents, patent applications and websites) that may be cited in this application are hereby expressly incorporated into the present disclosure in their entirety by reference, for any purpose. Unless otherwise indicated, the present disclosure may employ conventional techniques of microbiology, molecular biology and cell biology well known in the art.

[0103] The disclosed method and system may be modified without departing from the scope of this document.It should be noted that the matters shown in the above description or accompanying drawings should be interpreted as illustrative rather than restrictive.

Claims

1. A system for converting biomass into liquid fuel, comprising: a) Liquid reservoir; and b) a plurality of reactors arranged in series, each reactor having an inlet and an overflow outlet, the inlet of a first reactor of the plurality of reactors being connected to the liquid reservoir, wherein said plurality of reactors comprises a defined culture comprising at least one living organism capable of producing at least one carbohydrate active enzyme (CAZyme), wherein the steps of CAZyme production, substrate hydrolysis and fermentation by said defined culture without added enzymes all occur within each of said plurality of reactors.

2. The system of claim 1 , wherein overflow from the first reactor in the series is fed to a second reactor immediately downstream of the first reactor, and overflow from the Nth reactor is fed to an N+1th reactor immediately downstream of the Nth reactor, wherein N is an integer from 1 to n, and wherein n is an integer, and n+1 is equal to the total number of reactors in the plurality of reactors.

3. The system of claim 2, further comprising one or more mills positioned between two adjacent reactors.

4. The system of claim 2, further comprising a starting biomass, wherein the starting biomass is loaded into the reservoir, and the biomass flows unidirectionally from the reservoir to the first reactor.

5. The system of claim 1, wherein the plurality of reactors comprises 3 or more reactors.

6. The system of claim 1, wherein the system operates continuously or semi-continuously.

7. The system of claim 6, wherein the series of reactors operating continuously or semi-continuously shortens the reaction time from the biomass to the liquid fuel.

8. The system of claim 7, wherein the reaction time from the biomass to the liquid fuel is shorter than the reaction time in a batch or single-stage continuous bioreactor.

9. The system of claim 1, wherein the ratio between the CAZyme and the substrate in the first reactor is at least one unit of CAZyme per pmol of the substrate.

10. The system of claim 1, wherein the liquid fuel is ethanol.

11. The system of claim 1 , wherein the defined culture comprises Clostridium thermocellum and a hemicellulose fermenting microorganism selected from the group consisting of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium saccharolyticum.

12. The system of claim 11, wherein the starting biomass is one selected from the group consisting of corn stover, sugar cane, wheat straw or rice straw, miscanthus, switchgrass, energy cane, and combinations thereof.

13. A method of producing liquid fuel from biomass using the system of claim 1, comprising: a) loading the starting biomass into a reservoir, b) moving the biomass from the reservoir to a first reactor wherein at least a portion of the biomass is converted to alcohol, c) operating the system continuously or semi-continuously, wherein the liquid reservoir feeds the first reactor and overflow from the Nth reactor moves to the N+1th reactor immediately downstream of the Nth reactor, d) repeating the above steps (a)-(c) so that the biomass moves from the Nth reactor to the N+1 reactor through the reactors in series until it reaches the last reactor, e) operating the system continuously or semi-continuously, wherein the steps of CAZyme production, substrate hydrolysis and fermentation by the defined culture without added enzymes all occur within each of the reactors of the plurality of reactors, and f) collecting the liquid fuel from the plurality of reactors.

14. The method of claim 13, wherein the liquid fuel is ethanol.

15. The process of claim 13, wherein one or more mills placed between the reactor and an immediately downstream reactor mechanically treat the overflow.

16. The method of claim 14, wherein the system operates in a continuous or semi-continuous mode rather than in a batch mode.

17. The method of claim 13, wherein the solids loading is about 10-25 wt. % solids.

18. The method of claim 13, wherein the starting biomass is selected from the group consisting of corn stover, sugar cane, wheat straw or rice straw, miscanthus, switchgrass, energy cane, and combinations thereof.

19. The method of claim 13, wherein the ratio between the CAZyme and the substrate in the first reactor is at least one unit of CAZyme per pmol of the substrate.

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